This project defines how metabolism of nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS causes defective mitochondrial (mt-) DNA replication, oxidative stress and cardiomyopathy (CM, a weakness of the heart muscle with poor pump function). Along with the benefit of increased survival in HIV/AIDS, combinations that include NRTIs carry devastating mitochondrial side effects, including CM. Mitochondrial (mt-) DNA depletion and mutation, and oxidative stress are key mechanisms in CM and CHF, and for development of NRTI toxic mitochondrial side effects based of defective mtDNA replication. The working hypothesis states: In myocardial cells, NRTIs interact with proteins that control the nucleotide biosynthesis and salvage for mtDNA replication. Ribonucleotide reductase (RNR) and SAM domain and HD domain-containing protein 1 (SAMHD1) alter abundance of dNTPs for mtDNA replication. RNR inhibition or increased activity of SAMHD1 depletes native dNTP pools, increases abundance of NRTI-TPs (or both). mtDNA depletion and mutation, oxidative stress, and CM are the phenotypic signature. Through clinically relevant experiments using genetically engineered mice, the project will dissect nucleotide pool imbalance in the sarcoplasm and mitochondria of CM myocytes in HIV/AIDS. Oxidative stress and CM are ameliorated by protecting cardiac mitochondria with manganese porphyrin catalytic antioxidants that localize to mitochondria. AIM 1: To define the role of RNR activity and inhibition in modulating cardiac cytoplasmic and mitochondrial nucleotide pools in the CM of HIV/AIDS. Transgenic mice (TGs) that overexpress RNR are in the lab presently. In heart cells, RNR overexpression (by transgenesis) or its inhibition (e.g., exogenous resveratrol) alters nucleotide pools for phosphorylation and incorporation into mtDNA. Nucleotide pools are defined with dNTP assays and correlated with cardiac physiological, pathological and pharmacological studies. AIM 2: To define the role of SAMHD1 activity (and absence) in modulating the myocardial cytoplasmic and mitochondrial nucleotide pools in the CM of HIV/AIDS. SAMHD1 null mice are powerful biological tools to increase the pool of dNTPs in heart cells. Conversely, cardiac targeted transgenic overexpression of SAMHD1 increases dN pools. NRTI treatment generates NRTI-TPs that inhibit pol ? and create CM. Cardiac nucleotide pools are determined with dNTP assays and correlative physiological, pathological and pharmacological parameters. AIM 3: To prevent and treat CM in HIV/AIDS with replacement by ameliorating mitochondrial oxidative stress. Replacement of function is accomplished using TGs from AIM 1 and 2 that are co- administered manganese porphyrin catalytic antioxidants (to provide SOD2- and catalase-like activity that bolsters antioxidant defense) and prevent or attenuate RNR- or SAMHD1-induced cardiac changes and attenuate CM pathophysiological changes.